Much recent work has focused on the genes that encode the various contractile proteins. There is also an extensive literature focused on the behavior of individual purified contractile proteins in cell-free systems. Studies of the in vitro self-assembly of myosin, and of actin and actin-binding proteins continue to yield new information. However, little is known of how in living cells some 2 dozen of these proteins are sorted out spatially so that myofibrils are assembled in myogenic cells and stress fibers are assembled in non-muscle cells. To study the rules for the in vivo assembly of striated myofibrils or of stress fibers we selectively perturb one muscle-specific protein and then assess the consequences for the remaining contractile structures. For example, there is a sizeable literature claiming that desmin IFs play key roles in the assembly of Z-bands and in linking myofibrils to the sarcolemma. To test this we recently transfected myogenic cells with a truncated desmin cDNA. The polymerization of desmin into long IFs was totally blocked in the transfected cells. Nevertheless, these cells assembled normal, contracting striated myofibrils. Clearly the putative roles of desmin IFs during myofibrillogenesis have to be re-considered. We shall apply this novel technique of deletion mutagenesis and transfection, first to perturb individual contractile proteins and second, to follow the effects of such perturbations on the assembly of both myofibrils and stress fibers. Specifically we shall focus on the assembly of (a) sarcomeric alpha-actinin Z-bands and of non-sarcomeric alpha-actinin dense bodies, (b) sarcomeric MHC thick filaments in a A- bands and of non-sarcomeric MHC filaments in stress fibers, and 9c) alpha-actin thin filaments in I-bands and beta- and gamma- actin in stress fibers. In principle this should allow us to test, one-by-one, the role of each contractile protein in the assembly of either myofibrils or stress fibers.